Epothilone B Analogue (BMS-247550)-mediated Cytotoxicity through Induction of

[CANCER RESEARCH 62, 466 – 471, January 15, 2002]
Epothilone B Analogue (BMS-247550)-mediated Cytotoxicity through Induction of
Bax Conformational Change in Human Breast Cancer Cells1
Hirohito Yamaguchi, Shanthi R. Paranawithana, Michael W. Lee, Ziwei Huang, Kapil N. Bhalla, and
Hong-Gang Wang2
Drug Discovery Program [H. Y., M. W. L., H-G. W.] and Clinical Investigations Program [S. R. P., K. N. B.], H. Lee Moffitt Cancer Center and Research Institute, Tampa,
Florida 33612; Departments of Interdisciplinary Oncology [H. Y., S. R. P., K. N. B., H-G. W.] and Pharmacology [M. W. L., H-G. W.], University of South Florida, College of
Medicine, Tampa, Florida 33612; and Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 [Z. H.]
ABSTRACT
Epothilone B is a novel nontaxane antimicrotubule agent that is active
even against paclitaxel (Taxol)-resistant cancer cells. The present study
further explores the mechanisms underlying epothilone B-mediated cytotoxicity in human breast cancer cells. We show that BMS-247550 (EpoB),
a novel epothilone B analogue, induces cell cycle arrest at the G2-M phase
transition and subsequent apoptotic cell death of MDA-MB-468 (468)
cells. Treating cells with EpoB triggers a conformational change in the
Bax protein and its translocation from the cytosol to the mitochondria,
which is accompanied by cytochrome c release from the inter-membrane
space of mitochondria into the cytosol. Overexpression of Bcl-2 delays Bax
conformational change, cytochrome c release, and apoptosis induced by
EpoB. Conversely, the Bcl-2 antagonist Bak-BH3 peptide or HA14-1
compound abrogates the antiapoptotic effects of Bcl-2 and enhances apoptosis of 468 cells pretreated with EpoB (to induce mitotic arrest). In
synchronized 468 cells, EpoB is more potent in inducing Bax conformational change and apoptosis at G2-M phase compared with G1-S phase of
the cell cycle. Taken together, these findings demonstrate that EpoB
induces apoptosis through a Bcl-2-suppressible pathway that controls a
conformational change of the proapoptotic Bax protein. The enhanced
cytotoxicity of EpoB by blocking Bcl-2 at mitochondria implies a potential
application of the combination of EpoB and Bcl-2 antagonists in the
treatment of human breast cancer.
INTRODUCTION
Epothilone B is a novel nontaxane microtubule-targeting agent that
induces tubulin polymerization and stabilizes microtubules. Similar to
paclitaxel (Taxol), epothilone B also blocks the cell cycle at the G2-M
phase transition and subsequently induces apoptosis (1, 2). Although
Taxol is a highly effective drug against breast, ovarian, head and neck,
and non-small cell lung cancer (3, 4), epothilone B or its lactam
analogue BMS-247550 (EpoB) is more potent and active even against
Taxol-resistant cancer cells (1, 2, 5). However, the precise mechanisms underlying the cytotoxicity of these antimicrotubule agents
have yet to be elucidated.
Mitochondria play a central role in the signaling pathway for
apoptosis under many physiological and pathological conditions (6,
7). Cellular damage transduces intrinsic death signals to mitochondria,
resulting in the disruption of the mitochondrial potential (⌬␺) across
the inner membrane and leading to the release of several proapoptotic
molecules including cytochrome c, AIF, Smac, endonuclease G, and
HtrA2 into the cytosol (8 –12). Once released from mitochondria,
cytochrome c forms a multiprotein complex with Apaf-1 and procaspase-9, leading to activation of this initial caspase and induction of
downstream apoptotic protease cascade (13). The Bcl-2 family proteins are critical mediators of the mitochondrial pathway of apoptosis
Received 9/5/01; accepted 11/9/01.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported in part by Grants CA82197 and CA63382 from the NIH.
2
To whom requests for reprints should be addressed, at Drug Discovery Program, H.
Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL
33612.
that control the release of apoptogenic molecules from the mitochondria (14, 15). The antiapoptotic members of the Bcl-2 family such as
Bcl-2, Bcl-xL, and Mcl-1 prevent the release of cytochrome c from the
mitochondria, whereas the proapoptotic proteins like Bax, Bak, Bid,
Bad, and Bim promote it. Although the biochemical mechanism
underlying this process is still controversial, the relative ratios of proand antiapoptotic Bcl-2 family proteins decide the fate of the cell (16).
Bax is a proapoptotic member of the Bcl-2 protein family that is
predominantly localized in the cytosol of healthy cells and translocates to mitochondria after a variety of death stimuli (17). During
apoptosis, Bax undergoes a conformational change, leading to exposure of its N- and C-termini that appears to be required for the mitochondrial targeting of the Bax protein (17, 18). Once translocated to the outer
membranes of the mitochondria, Bax forms oligomers or clusters that
cause mitochondrial dysfunction and apoptotic cell death (19, 20). The
proapoptotic Bcl-2 family member Bid promotes the mitochondrial translocation of Bax, whereas the antiapoptotic proteins Bcl-2 and Bcl-xL
inhibit Bax conformational change and mitochondria-associated Bax
cluster formation (20, 21). Thus, Bax conformational change is a critical
step in the Bax-mediated signaling pathway for apoptosis.
In this paper, we investigated the role of Bax and Bcl-2 in the novel
epothilone B analogue BMS-247550 (EpoB)-induced apoptosis of
human breast cancer cells. We found that EpoB triggers a conformational change in the NH2 terminus of the Bax protein and subsequently induces apoptotic cell death in MDA-MB-468 cells through a
Bcl-2-suppressible mechanism.
MATERIALS AND METHODS
Materials. BMS-247550 (EpoB) was kindly provided by Bristol-Myers
Squibb (Princeton, NJ). Anti-Bax 6A7 monoclonal antibody was purchased
from Sigma Chemical Co. (St. Louis, MO). Anti-Bid polyclonal antibody,
anti-cytochrome c monoclonal antibody, and anti-Bcl-2 4D7 monoclonal antibody were purchased from PharMingen (San Diego, CA). Anti-COX IV
monoclonal antibody was purchased from Molecular Probes (Eugene, OR).
Antihuman Bax, Bcl-2, and Bcl-xL polyclonal antibodies were described
previously (22). Cellular membrane-permeable Bak-BH3 peptide, and monoclonal antibodies against caspase-3 and cleaved D4-GDI were kindly provided
by Imgenex (San Diego, CA). Caspase inhibitor Z-VAD-FMK was purchased
from Alexis (San Diego, CA). Bcl-2 inhibitor HA14-1 was described previously (23). Human breast cancer cell line MDA-MB-468 (468) was obtained
from the American Type Culture Collection (Manassas, VA).
Cell Culture and Transfection. The 468 cells were maintained in RPMI
1640 medium supplemented with 10% FCS and 1% penicillin-streptomycin.
To establish Bcl-2-stable transfectants, pRC/CMV-Bcl-2 plasmid DNA was
transfected into 468 cells by TRANSIT transfection reagent (PanVera, Madison, WI), and positive clones were selected with 1 mg/ml G418.
MTT Assay. Cells were seeded at 2 ⫻ 104 cells per well of 96-well plates
in 0.1 ml of complete medium and cultured for 12 h. At the indicated time
points after drug treatment, 10 ␮l of 5 mg/ml MTT3 solution (MTT Thiazolyl
blue) was added into the cell cultures and incubated for 3 h at 37°C. The
reaction was stopped by adding 0.1 ml of 10% SDS in 10 mM HCl. After an
3
The abbreviations used are: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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EPOTHILONE B INDUCES BAX CONFORMATIONAL CHANGE
additional incubation at 37°C for 12 h, the absorbance at 570 nm was measured
using a microplate reader, and the cell viability was expressed as percentage
relative to time 0.
DNA Fragmentation Assay. Cells in 6-cm culture dishes were harvested
and incubated in 100 ␮l of lysis buffer (100 mM NaCl, 100 mM Tris-HCl, pH
8.0, 25 mM EDTA, 0.5% SDS, 100 ␮g/ml proteinase K) at 50°C overnight,
followed by ethanol precipitation. The pellets were dissolved in TE buffer (10
mM Tris-HCl, pH 8.0, 1 mM EDTA) containing 1 ␮g/ml RNase A and
incubated for 1 h at 37°C. The DNA samples were subjected to 1.5% agarose
gel and visualized with ethidium bromide staining.
Detection of Bax Conformational Change. Cells were lysed with Chaps
lysis buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 1% Chaps) containing
protease inhibitors as described (24). The cell lysates were normalized for
protein content and 500 ␮g of total protein were incubated with 2 ␮g of
anti-Bax 6A7 monoclonal antibody in 500 ␮l of Chaps lysis buffer for 3 h at
4°C. Then, 25 ␮l of protein G-agarose were added into the reactions and
incubated at 4°C for an additional 2 h. After three washings in Chaps lysis
buffer, beads were boiled in Laemmli buffer, and the conformationally
changed Bax protein in the immunoprecipitates was subjected to SDS-PAGE
immunoblot analysis with anti-Bax polyclonal antibody as described (24).
Subcellular Fractionation. Cells were harvested and resuspended in 3
volumes of isotonic lysis buffer (210 mM sucrose, 70 mM mannitol, 10 mM
HEPES, pH7.4, 1 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride,
5 ␮g/ml leupeptin, 5 ␮g/ml aprotinin, and 0.7 ␮g/ml pepstatin. After homogenization with a Dounce homogenizer, cell lysates were centrifuged at
1,000 ⫻ g to discard nuclei. The postnuclear supernatant was centrifuged at
10,000 ⫻ g to pellet the mitochondria-enriched heavy membrane fraction. The
supernatant was further centrifuged at 100,000 ⫻ g to obtain cytosolic fraction.
The membrane fractions were resuspended in Triton X-100 lysis buffer containing protease inhibitors (24). Protein concentration was determined by BCA
assay (Pierce Chemical, Rockford, IL).
Cell Cycle Synchronization. Cells were treated with 2 mM thymidine for
17 h to synchronize the cell cycle at early S phase. After washing three times
in RPMI 1640 medium, cells were shifted to complete medium to release the
thymidine block. Cell cycle status was determined by DNA content analysis of
propidium iodide-stained cells with a Becton Dickinson FACSort flow cytometer as described previously (22).
RESULTS
EpoB Induces Mitotic Arrest and Apoptosis of Human Breast
Cancer Cells. BMS-247550 (EpoB) is a lactam analogue of epothilone B developed by Bristol-Myers Squibb, which is highly active in
vitro and in vivo even against human cancers that are resistant to
paclitaxel (5). To further understand the mechanisms of the antineoplastic effects of epothilone B, human breast cancer cells MDAMB-468 (468) were treated with different concentrations of EpoB for
24 h or with 50 nM EpoB for various periods. The cell viability was
assessed by MTT assay. As shown in Fig. 1, A and B, EpoB induced
cell death of 468 cells in a dose- and time-dependent manner. To
confirm that this cell death was apoptosis, we examined DNA ladder
formation (one of the characteristics of apoptosis) in EpoB-treated
468 cells. A clear DNA fragmentation was observed at 24 h after
treatment with 50 nM EpoB, indicating that EpoB induces apoptotic
cell death in these human breast cancer cells (Fig. 1C). Apoptosis is
caused by the activation of a family of cysteine-dependent aspartatespecific proteases known as caspases. Thus, we examined the activation of caspase-3, an executioner caspase, in 468 cells exposed to 50
nM EpoB. Caspase-3 activation was evaluated by immunoblot analysis
with anti-caspase-3 monoclonal antibody that recognizes both proand activated (p20 and p17) caspase-3. As shown in Fig. 1D,
caspase-3 was apparently activated around 18 h after EpoB treatment.
Cleavage of D4-GDI (a caspase-3 substrate) demonstrated further
caspase-3 activation within 468 cells treated with EpoB.
It has been shown that epothilone B has an action similar to that of
Taxol, which binds to and stabilizes microtubules leading to mitotic
arrest of the cell cycle and subsequent apoptosis in several cell lines
(1, 2). To determine whether EpoB induces mitotic arrest in human
breast cancer cells, we performed cell cycle analysis of 468 cells after
EpoB treatment. As summarized in Fig. 1E, exposure to 50 nM EpoB
triggered cells to accumulate in G2-M phase of the cell cycle, beginning at 8 h and reaching a maximum at 24 h after treatment. The
population of sub-G1 cells (apoptotic cells) was significantly increased
Fig. 1. EpoB treatment causes G2-M phase arrest and
subsequently induces apoptosis in 468 cells. A, 468 cells
were treated with different concentrations of EpoB for
24 h, and cell viability was evaluated by MTT assay
(mean ⫾ SD; n ⫽ 3). B, 468 cells were treated with 50 nM
EpoB for various periods, and cell viability was determined by MTT assay (mean ⫾ SD; n ⫽ 3). C, 468 cells
were treated with 50 nM EpoB, and DNA ladder was
detected as described in “Materials and Methods.” D, 468
cells were treated with 50 nM EpoB for 0, 12, 18, or 24 h,
followed by preparation of cell lysates and SDS-PAGE/
immunoblot assay with monoclonal antibodies specific
for caspase-3, cleaved D4-GDI, or ␣-tubulin as control. E,
468 cells were treated with 50 nM EpoB and subjected to
DNA content analysis by flow cytometry at the indicated
time points.
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EPOTHILONE B INDUCES BAX CONFORMATIONAL CHANGE
at 24 h after EpoB treatment, suggesting that EpoB blocks cell cycles at
the G2-M phase and subsequently induces apoptosis in 468 cells.
EpoB Induces Bax Conformational Change and Mitochondrial
Translocation. Because the Bcl-2 family proteins are known to regulate the mitochondrial pathway of apoptosis, we next examined the
expression of several Bcl-2 family proteins in 468 cells treated with
EpoB. Immunoblotting analysis revealed that the protein levels of
Bcl-2, Bcl-xL, Bax, and Bid were not significantly affected by 50 nM
EpoB treatment for up to 24 h (Fig. 2A), although a slight proportion
of Bid was cleaved after 18 h of treatment (Fig. 2A). In addition,
treating 468 cells with EpoB slightly induced a mobility shift of
endogenous Bcl-2 protein in SDS-polyacrylamide gels (Fig. 2A),
similar to those observed in Taxol-treated cells (25), indicating that
Bcl-2 is phosphorylated by EpoB treatment.
It has been reported that Bax undergoes a conformational change
and translocation to mitochondria during apoptosis (17, 26). To
examine Bax conformation, 468 cells were lysed in 1% Chaps
(a condition that retains the Bax protein in its native conformation),
and immunoprecipitation was performed with anti-Bax 6A7 monoclonal antibody that only recognizes conformationally changed Bax
(24, 27). As shown in Fig. 2B, conformationally changed Bax was
Fig. 2. EpoB induces Bax conformational change and cytochrome c release. A, 468
cells were treated with 50 nM EpoB for various times and subjected to SDS-PAGE/
immunoblot analysis with the indicated antibodies. B, 468 cells were treated with 50 nM
EpoB and applied to immunoprecipitation with anti-Bax 6A7 antibody for detection of
conformationally changed Bax protein. C, 468 cells were exposed to 50 nM EpoB for 0,
12, or 24 h and subjected to subcellular fractionation. The mitochondria-enriched heavy
membrane fraction (Mitochondria) and cytosolic fraction (Cytosol) were analyzed by
immunoblotting (100 ␮g of protein per lane) with antibodies specific for Bax, cytochrome
c, or mitochondrial protein COX IV for control. D, 468 cells were treated with 50 nM
EpoB in the presence (⫹) or absence (⫺) of 100 ␮M Z-VAD-FMK (z-VAD) for 24 h. Cell
lysates were prepared in Chaps lysis buffer and subjected to immunoprecipitation with
anti-Bax 6A7 antibody or applied directly to SDS-PAGE/immunoblot analysis with
anti-Bid or anti-Bax antibody. E, 468 cells were incubated with 50 nM EpoB for 0 or 24 h
before preparation of cell lysates in Chaps lysis buffer. Immunoprecipitation was performed using anti-Bax polyclonal serum, followed by SDS-PAGE/immunoblot analysis
with antibodies specific for Bax or Bif-1. In addition to immune complexes, the total
lysates (30 ␮g of protein per lane) were analyzed directly.
detectable in 468 cells from 12 h after treatment with 50 nM EpoB,
indicating that EpoB could induce Bax conformational change after
mitotic arrest of the cell cycle. Moreover, subcellular fractionation
experiments (Fig. 2C) revealed that the protein level of Bax decreased
in the cytosolic fraction and increased in the mitochondria-enriched
heavy membrane fraction of 468 cells during EpoB treatment. This
indicates that EpoB induces Bax translocation from the cytosol to the
mitochondria. In addition, immunoblot analysis of the fractionated
cell lysates with anti-cytochrome c antibody showed that cytochrome
c was released from the mitochondria into the cytosol at 24 h after
EpoB treatment (Fig. 2C). The mitochondrial protein COX IV was
used as a control for the fractionation procedure.
Bid is a BH3-only proapoptotic member of the Bcl-2 family that
undergoes proteolysis during apoptosis (28, 29), and the resulting
truncated Bid (tBid) induces Bax conformational change and subsequent translocation to mitochondria (26, 30). Therefore, we examined
the role of Bid cleavage in EpoB-induced Bax conformational change.
Treating 468 cells with the caspase inhibitor Z-VAD-FMK blocked
Bid cleavage (Fig. 2D) and apoptosis (not shown), but had no effect
on Bax conformational change (Fig. 2D) induced by EpoB. These
results suggest that Bax conformational change is an early event
upstream of caspase activation and Bid cleavage in EpoB-mediated
apoptosis.
Bif-1 is a novel Bax-binding protein that promotes interleukin-3
deprivation-induced Bax conformational change and apoptotic cell
death, when overexpressed in murine pre-B hematopoietic FL5.12
cells (24). Interleukin-3 withdrawal from cultured FL5.12 cells results
in increased association of Bax with Bif-1, which may play a role in
subsequent Bax conformational change (24). Consistent with this,
coimmunoprecipitation analysis revealed that EpoB also induced Bax
association with Bif-1 in 468 cells (Fig. 2E), implying that Bif-1 may
be involved in EpoB-mediated Bax conformational change.
Bcl-2 Decreases EpoB-induced Bax Conformational Change
and Apoptosis. Overexpression of Bcl-2 protects cells from apoptosis induced by multiple chemotherapeutic drugs (31), but it still
remains controversial whether Bcl-2 blocks cell death induced by
microtubule-damaging agents including Taxol (25, 32–34). To determine the effect of Bcl-2 on EpoB-induced apoptosis, we stably transfected 468 cells with human Bcl-2. Several independent clones of 468
transfectants with a variety of Bcl-2 expression levels were obtained
and used to determine the relationship between Bcl-2 expression and
EpoB resistance (Fig. 3A). As shown in Fig. 3, B and C, Bcl-2
significantly delayed EpoB-induced apoptosis of 468 cells, which had
a correlation with the higher expression levels of this antiapoptotic
protein. The inhibitory effect of Bcl-2 against EpoB-mediated cytotoxicity was comparable with that observed in cells treated with
etoposide or staurosporine (Fig. 3C). However, overexpression of
Bcl-2 in 468 cells had no significant effect on EpoB-induced mitotic
arrest of the cell cycle (not shown).
To elucidate the mechanism underlying Bcl-2 protection against
EpoB-induced apoptosis, we determined the ability of Bcl-2 to retain
Bax conformation after EpoB treatment. The 468/Bcl-2 or control 468
cells were treated with 50 nM EpoB for various times before preparation of cell lysates in 1% Chaps. The conformationally changed Bax
protein was detected by immunoprecipitation with anti-Bax 6A7
monoclonal antibody. Compared with control cells, overexpression of
Bcl-2 apparently delayed Bax conformational change after EpoB
treatment (Fig. 4A). In addition, subcellular fractionation analysis
revealed that Bax translocation to mitochondria and cytochrome c
release were also delayed in 468 cells overexpressing Bcl-2 compared
with control cells after EpoB treatment (Fig. 4B).
Inhibition of Bcl-2 Enhances the Cytotoxicity of EpoB. If Bcl-2
has a pivotal role in the EpoB-mediated mitochondrial pathway for
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EPOTHILONE B INDUCES BAX CONFORMATIONAL CHANGE
Bcl-2 protected 468 cells from EpoB-induced apoptosis throughout
the cell cycle, but it was less effective in G2-M cells (Fig. 6B). In
contrast, Bak-BH3 peptide killed 468 cells in a cell cycle-independent
manner (Fig. 6C). Furthermore, immunoprecipitation experiments
with anti-Bax 6A7 antibody showed that EpoB rapidly induced Bax
conformational change in cells that were released from thymidine
block for 6 h (at this time, a majority of cells were in G2-M phase)
compared with G1-S phase (time 0) cells (Fig. 6D).
DISCUSSION
Fig. 3. Bcl-2 protects 468 cells from apoptosis induced by EpoB. A, 468 cells were
transfected with plasmid DNA encoding human Bcl-2 protein and selected by G418 to
obtain stable clones. Three independent clones (#5, #6, and #7) and control 468 (C) cells
were analyzed by SDS-PAGE/immunoblotting with antibodies specific for Bcl-2 or
control protein ␣-tubulin. B, stably transfected 468 clones were treated with 50 nM EpoB,
and cell viability was determined 2 days later by MTT assay (mean ⫾ SD; n ⫽ 3). C,
468/Bcl-2 (clone #6) and control 468 (C) cells were treated with either 50 nM EpoB, 12.5
␮g/ml etoposide, or 1 ␮M staurosporine (STS) for 24 or 48 h, and cell viability was
evaluated by MTT assay (mean ⫾ SD; n ⫽ 3).
apoptosis, blockade of Bcl-2 should sensitize cells to apoptosis induced by EpoB. It has been shown that the membrane-permeable
Bak-BH3 peptide (fused to the internalization domain of the Antennapedia homeoprotein) as well as the small compound HA14-1 kills
cells through binding to and antagonizing the antiapoptotic proteins
Bcl-2 and Bcl-xL (23, 35). Thus, we treated 468 cells with EpoB
and/or Bcl-2 inhibitor Bak-BH3 peptide or HA14-1. As shown in Fig.
5, treatment with either Bak-BH3 peptide or HA14-1 induced cell
death in 468 cells, indicating that Bcl-2/Bcl-xL is essential for the
survival of this human breast cancer line. Moreover, these Bcl-2
antagonists could overcome the cytoprotective effects of the overexpressed Bcl-2 protein (Fig. 5). When EpoB was added to the cell
cultures with the Bcl-2 inhibitors at the same time, there was no
additive effect of EpoB on apoptosis induced by Bak-BH3 peptide or
HA14-1 (Fig. 5A). On the contrary, when the cells were exposed to
Bcl-2 inhibitors after pretreatment with EpoB for 12 h, an additive or
supra-additive effect of the Bcl-2 inhibitors on EpoB-induced cell
death was observed in both 468/Bcl-2 and control 468 cells (Fig. 5B).
EpoB Is More Active in Inducing Bax Conformational Change
and Apoptosis in G2-M Phase Cells. To determine whether the
EpoB-mediated cytotoxic effect is cell cycle dependent, we synchronized 468/Bcl-2 and control 468 cells in early S phase of the cell cycle
by thymidine treatment. DNA content analysis revealed that cells
were in S phase (85–91%), G2-M phase (58 – 65%), and G1 phase
(67%) at 2, 6, and 10 h, respectively, after release of cells from
thymidine block (Fig. 6A). At various times after release from thymidine block, cells were treated with 50 nM EpoB for 12 h, and the
cell viability was determined by MTT assay. As shown in Fig. 6B, a
maximal cytotoxicity of EpoB was observed at 4 – 6 h after thymidine
release, when ⬃65% of cells were in G2-M phase of the cell cycle.
The findings reported here demonstrate that BMS-247550 (EpoB),
a lactam analogue of epothilone B, kills cells through a mitochondrial
pathway of apoptosis controlled by Bcl-2 and Bax. Similar to Taxol,
EpoB causes cell cycle arrest at the G2-M transition and secondarily
induces apoptosis of human breast cancer cells. In agreement with
this, cells synchronized in G2-M phase of the cell cycle are more
sensitive to EpoB-induced cytotoxicity. Upon EpoB treatment, Bax
undergoes a conformational change and subsequently translocates to
mitochondria. This conformational change of Bax occurs upstream
of Bid cleavage, cytochrome c release, and caspase activation. In
addition, overexpression of Bcl-2 decreases EpoB-induced Bax conformational change, cytochrome c release, and apoptosis, whereas
blockade of Bcl-2 by Bak-BH3 peptide or HA14-1 increases EpoBmediated cytotoxicity.
In healthy cells, Bax exists predominantly in the cytosol, despite the
presence of a typical membrane-anchoring sequence near its COOHterminus (17, 36). It has been reported that the putative transmembrane domain is hidden in the inactive cytosolic form of Bax and
becomes accessible to mitochondrial targeting following apoptotic
signals (17, 37). Early during apoptosis, Bax undergoes a conformational change and translocates to mitochondria where it participates in
Fig. 4. Bcl-2 delays EpoB-induced Bax conformational change and cytochrome c
release. The 468/Bcl-2 (clone #6) and control 468 (C) cells were treated with 50 nM EpoB
for various times and subjected to the following analysis. A, cells were lysed in Chaps
lysis buffer, and the conformationally changed Bax was detected by immunoprecipitation
with anti-Bax 6A7 antibody. B, subcellular fractionation was performed to produce
cytosolic (Cytosol) and heavy membrane (Mitochondria) fractions, which were subjected
to SDS-PAGE/immunoblot analysis (100 ␮g of protein per lane) with the indicated
antibodies.
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EPOTHILONE B INDUCES BAX CONFORMATIONAL CHANGE
Fig. 5. Blockade of Bcl-2 enhances EpoB-induced apoptosis. A,
468/Bcl-2 (clone #6) and control 468 (C) cells were treated with
either 50 nM EpoB, 50 ␮M Bak-BH3 peptide, or 30 ␮M HA14-1
alone, or with a combination of 50 nM EpoB and 50 ␮M Bak-BH3
peptide or 30 ␮M HA14-1 for 16 h. Cell viability was evaluated by
MTT assay (mean ⫾ SD; n ⫽ 3). B, 468/Bcl-2 (clone #6) and
control 468 (C) cells were cultured with 50 nM EpoB or control
DMSO for 12 h and subsequently treated with 50 ␮M Bak-BH3
peptide or 30 ␮M HA14-1 for an additional 8 h. Cell viability was
determined by MTT assay (mean ⫾ SD; n ⫽ 3).
mitochondrial disruption and the release of cytochrome c (6, 38).
Although exactly how mitochondrial apoptogenic molecules escape
during apoptosis remains unclear, one possibility is that Bax may form
selective channels for cytochrome c release from the inter-membrane
space of mitochondria into the cytosol (6).
The mechanism by which EpoB treatment causes a conformational
change of the Bax protein remains to be elucidated. It is possible that
EpoB induces a rapid increase in intracellular pH, which has been
suggested to trigger Bax conformational change and subsequent translocation to mitochondria (39). In addition, some factor(s) may become
activated after mitotic arrest induced by EpoB, which directly triggers
Bax conformational change. One of the potential candidates is the
proapoptotic protein Bid, which undergoes proteolysis during apoptosis, and the cleaved product (tBid) promotes Bax conformational
change (26, 30). Although Bid has been shown to be important for
Bax conformational change induced by extrinsic death signals such as
Fas and tumor necrosis factor ␣ (26, 40), it is not required for cellular
damage (intrinsic death signal)-mediated conformational change of
the Bax protein (41, 42). Consistent with this, our results indicate that
Bid cleavage is not necessary for EpoB-induced Bax conformational
change in human breast cancer cells. Another potential activator for
Bax conformational change is Bif-1, which binds to native/inactive
Bax during apoptosis that may have a role in the Bax-mediated
pathway of apoptosis (24). Indeed, treating cells with EpoB induces
increased association between Bax and Bif-1. Moreover, some unidentified factors may exist in mitochondria, because Bcl-2 prevents
Bax conformational change that is mainly localized at the outer
membranes of mitochondria. This is supported by our results that
blocking of Bcl-2 by Bak-BH3 peptide or HA14-1, which directly
binds to and antagonizes the antiapoptotic protein Bcl-2/Bcl-xL (23,
35), promotes apoptosis induced by EpoB. In addition, the BH3-only
proapoptotic protein Bim has recently been shown to function
upstream of Bax-mediated cytochrome c release (43). It will be
interesting to see whether EpoB induces Bim translocation from
microtubules to mitochondria where it binds to and abrogates the
antiapoptotic activity of Bcl-2, thus promoting Bax conformational
change and inducing apoptosis.
Fig. 6. The ability of EpoB to induce apoptosis and Bax conformational change is
G2-M dependent. 468/Bcl-2 (clone #6) and control 468 (C) cells were incubated with 2
mM thymidine for 17 h to synchronize cells in early S phase. A, cell cycle status was
determined by DNA content analysis at various time points after the release of cells from
thymidine block. B, synchronized cells were cultured in fresh medium to release thymidine block for various times and then treated with 50 nM EpoB for 12 h. The cell viability
was evaluated by MTT assay (mean ⫾ SD; n ⫽ 3). C, synchronized cells were released
from thymidine block for 0 or 6 h before treatment with 50 nM EpoB or 50 ␮M Bak-BH3
peptide for 6 or 12 h, followed by MTT assay (mean ⫾ SD; n ⫽ 3). D, after release of
cells from thymidine block for 0 or 6 h, cells were treated with 50 nM EpoB for 6 h and
subjected to immunoprecipitation with anti-Bax 6A7 antibody to detect conformationally
changed Bax protein. In addition to immune complexes, total lysates were also applied
directly to SDS-PAGE/immunoblot analysis.
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EPOTHILONE B INDUCES BAX CONFORMATIONAL CHANGE
It has been proposed that the mechanism by which Taxol exerts its
cytotoxic effect involves mitotic arrest, resulting in JNK activation
and Bcl-2 phosphorylation and inactivation leading to apoptosis (44).
EpoB also causes a slower migration of the Bcl-2 protein in gels,
similar to what occurred in Taxol-treated cells (not shown), indicating
a post-translational modification of Bcl-2. However, Bcl-2 can significantly delay EpoB-induced apoptosis when overexpressed in
human breast cancer cells, suggesting that this post-translational modification may not have an essential effect on the cytoprotective activity of Bcl-2. Until further evidence is provided, we do not exclude the
possibility that EpoB-mediated phosphorylation of Bcl-2 does inactivate this antiapoptotic protein, because there is a significant proportion of Bcl-2 without post-translational modification in Bcl-2 transfected cells after EpoB treatment (not shown).
High levels of bcl-2 gene expression have been documented in a
wide variety of human cancer (31). Overexpression of Bcl-2 contributes not only to the origins of cancer but also to treatment failures,
because the relative levels of Bcl-2 protein control the sensitivity of
tumor cells to cell death induced by nearly all chemotherapeutic drugs
as well as radiation (31). Thus, inhibition of Bcl-2 may put tumor cells
into a more vulnerable state with reference to apoptosis induction by
currently available anticancer drugs. The present study demonstrates
that the Bcl-2 inhibitor Bak-BH3 peptide or HA14-1 enhances EpoBinduced cell death of human breast cancer cells. These findings may
lead to the development of additional supplementary and even synergistic strategies for treating breast cancer patients.
ACKNOWLEDGMENTS
We thank Dr. Itaru Hirai for helpful discussion, Dr. Chun Wu (Imgenex
Corp., San Diego, CA) for providing Bak-BH3 peptide, Bristol-Myers Squibb
(Princeton, NJ) for providing BMS-247550 (EpoB), and the members of Flow
Cytometry Core at the Moffitt Cancer Center for assistance.
REFERENCES
1. Bollag, D. M., McQueney, P. A., Zhu, J., Hensens, O., Koupal, L., Liesch, J., Goetz, M.,
Lazarides, E., and Woods, C. M. Epothilones, a new class of microtubule-stabilizing
agents with a Taxol-like mechanism of action. Cancer Res., 55: 2325–2333, 1995.
2. Kowalski, R. J., Giannakakou, P., and Hamel, E. Activities of the microtubulestabilizing agents epothilones A and B with purified tubulin and in cells resistant to
paclitaxel (TaxolR). J. Biol. Chem., 272: 2534 –2541, 1997.
3. Rowinsky, E. K., and Donehower, R. C. Paclitaxel (Taxol). N. Engl. J. Med., 332:
1004 –1014, 1995.
4. Crown, J., and O’Leary, M. The taxanes: an update. Lancet, 355: 1176 –1178, 2000.
5. Lee, F. Y., Borzilleri, R., Fairchild, C. R., Kim, S. H., Long, B. H., Reventos-Suarez,
C., Vite, G. D., Rose, W. C., and Kramer, R. A. BMS-247550: a novel epothilone
analog with a mode of action similar to paclitaxel but possessing superior antitumor
efficacy. Clin. Cancer Res., 7: 1429 –1437, 2001.
6. Green, D. R., and Reed, J. C. Mitochondria and apoptosis. Science (Wash. DC), 281:
1309 –1312, 1998.
7. Desagher, S., and Martinou, J. C. Mitochondria as the central control point of
apoptosis. Trends Cell Biol., 10: 369 –377, 2000.
8. Liu, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. Induction of apoptotic
program in cell-free extracts: requirement for dATP and cytochrome c. Cell, 86:
147–157, 1996.
9. Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Snow, B. E., Brothers, G. M.,
Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., et al. Molecular characterization
of mitochondrial apoptosis-inducing factor. Nature (Lond.), 397: 441– 446, 1999.
10. Du, C., Fang, M., Li, Y., Li, L., and Wang, X. Smac, a mitochondrial protein that
promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition.
Cell, 102: 33– 42, 2000.
11. Li, L. Y., Luo, X., and Wang, X. Endonuclease G is an apoptotic DNase when
released from mitochondria. Nature (Lond.), 412: 95–99, 2001.
12. Suzuki, Y., Imai, Y., Nakayama, H., Takahashi, K., Takio, K., and Takahashi, R. A
serine protease, htra2, is released from the mitochondria and interacts with xiap,
inducing cell death. Mol. Cell, 8: 613– 621, 2001.
13. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S.,
and Wang, X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9
complex initiates an apoptotic protease cascade. Cell, 91: 479 – 489, 1997.
14. Wang, H-G., and Reed, J. C. Mechanisms of Bcl-2 protein function. Histol. Histopathol., 13: 521–530, 1998.
15. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, I-I., Jones,
D. P., and Wang, X. Prevention of apoptosis by Bcl-2: release of cytochrome c from
mitochondria blocked. Science (Wash. DC), 275: 1129 –1132, 1997.
16. Oltvai, Z. N., and Korsmeyer, S. J. Checkpoints of dueling dimers foil death wishes.
Cell, 79: 189 –192, 1994.
17. Wolter, K. G., Hsu, Y. T., Smith, C. L., Nechushtan, A., Xi, X. G., and Youle, R. J.
Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol.,
139: 1281–1292, 1997.
18. Nechushtan, A., Smith, C. L., Hsu, Y. T., and Youle, R. J. Conformation of the Bax
C-terminus regulates subcellular location and cell death. EMBO J., 18: 2330 –2341, 1999.
19. Gross, A., Jockel, J., Wei, M. C., and Korsmeyer, S. J. Enforced dimerization of BAX
results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J., 17:
3878 –3885, 1998.
20. Nechushtan, A., Smith, C. L., Lamensdorf, I., Yoon, S. H., and Youle, R. J. Bax and
Bak coalesce into novel mitochondria-associated clusters during apoptosis. J. Cell
Biol., 153: 1265–1276, 2001.
21. Eskes, R., Desagher, S., Antonsson, B., and Martinou, J. C. Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol. Cell. Biol.,
20: 929 –935, 2000.
22. Komatsu, K., Miyashita, T., Hang, H., Hopkins, K. M., Zheng, W., Cuddeback, S.,
Yamada, M., Lieberman, H. B., and Wang, H. G. Human homologue of S. pombe Rad9
interacts with BCL-2/BCL-xL and promotes apoptosis. Nat. Cell Biol., 2: 1– 6, 2000.
23. Wang, J. L., Liu, D., Zhang, Z. J., Shan, S., Han, X., Srinivasula, S. M., Croce, C. M.,
Alnemri, E. S., and Huang, Z. Structure-based discovery of an organic compound that
binds Bcl-2 protein and induces apoptosis of tumor cells. Proc. Natl. Acad. Sci. USA,
97: 7124 –7129, 2000.
24. Cuddeback, S. M., Yamaguchi, H., Komatsu, K., Miyashita, T., Yamada, M., Wu, C.,
Singh, S., and Wang, H. G. Molecular cloning and characterization of Bif-1: a novel
SH3 domain-containing protein that associates with Bax. J. Biol. Chem., 276:
20559 –20565, 2001.
25. Haldar, S., Jena, N., and Croce, C. M. Inactivation of Bcl-2 by phosphorylation. Proc.
Natl. Acad. Sci. USA, 92: 4507– 4511, 1995.
26. Murphy, K. M., Streips, U. N., and Lock, R. B. Bcl-2 inhibits a Fas-induced
conformational change in the Bax N-terminus and Bax mitochondrial translocation.
J. Biol. Chem., 275: 17225–17228, 2000.
27. Hsu, Y. T., and Youle, R. J. Bax in murine thymus is a soluble monomeric protein that
displays differential detergent-induced conformations. J. Biol. Chem., 273: 10777–
10783, 1998.
28. Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. Bid, a Bcl2 interacting
protein, mediates cytochrome c release from mitochondria in response to activation of
cell surface death receptors. Cell, 94: 481– 490, 1998.
29. Li, H., Zhu, H., Xu, C. J., and Yuan, J. Cleavage of BID by caspase 8 mediates the
mitochondrial damage in the Fas pathway of apoptosis. Cell, 94: 491–501, 1998.
30. Desagher, S., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S.,
Maundrell, K., Antonsson, B., and Martinou, J. C. Bid-induced conformational
change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol., 144: 891–901, 1999.
31. Reed, J. C. Bcl-2: prevention of apoptosis as a mechanism of drug resistance.
Hematol.-Oncol. Clin. N. Am., 9: 451– 474, 1995.
32. Ibrado, A. M., Liu, L., and Bhalla, K. Bcl-xL overexpression inhibits progression of
molecular events leading to paclitaxel-induced apoptosis of human acute myeloid
leukemia HL-60 cells. Cancer Res., 57: 1109 –1115, 1997.
33. Fang, G., Chang, B. S., Kim, C. N., Perkins, C., Thompson, C. B., and Bhalla, K. N.
“Loop” domain is necessary for Taxol-induced mobility shift and phosphorylation of
Bcl-2 as well as for inhibiting Taxol-induced cytosolic accumulation of cytochrome
c and apoptosis. Cancer Res., 58: 3202–3208, 1998.
34. Yamamoto, K., Ichijo, H., and Korsmeyer, S. J. BCL-2 is phosphorylated and
inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at
G2/M. Mol. Cell Biol., 19: 8469 – 8478, 1999.
35. Holinger, E. P., Chittenden, T., and Lutz, R. J. Bak BH3 peptides antagonize Bcl-xL
function and induce apoptosis through cytochrome c-independent activation of
caspases. J. Biol. Chem., 274: 13298 –13304, 1999.
36. Hsu, Y-T., Wolter, K. G., and Youle, R. J. Cytosol-to-membrane redistribution of
members of Bax and Bcl-XL during apoptosis. Proc. Natl. Acad. Sci. USA, 94:
3668 –3672, 1997.
37. Suzuki, M., Youle, R. J., and Tjandra, N. Structure of bax. Coregulation of dimer
formation and intracellular localization. Cell, 103: 645– 654, 2000.
38. Roucou, X., and Martinou, J. C. Conformational change of Bax: a question of life or
death. Cell Death Differ., 8: 875– 877, 2001.
39. Khaled, A. R., Kim, K., Hofmeister, R., Muegge, K., and Durum, S. K. Withdrawal
of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in
intracellular pH. Proc. Natl. Acad. Sci. USA, 96: 14476 –14481, 1999.
40. Perez, D., and White, E. TNF-␣ signals apoptosis through a bid-dependent conformational change in Bax that is inhibited by E1B 19K. Mol. Cell, 6: 53– 63, 2000.
41. Perkins, C. L., Fang, G., Kim, C. N., and Bhalla, K. N. The role of Apaf-1, caspase-9,
and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during
apoptosis. Cancer Res., 60: 1645–1653, 2000.
42. Wei, M. C., Zong, W. X., Cheng, E. H., Lindsten, T., Panoutsakopoulou, V., Ross,
A. J., Roth, K. A., MacGregor, G. R., Thompson, C. B., and Korsmeyer, S. J.
Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and
death. Science (Wash. DC), 292: 727–730, 2001.
43. Putcha, G. V., Moulder, K. L., Golden, J. P., Bouillet, P., Adams, J. A., Strasser, A.,
and Johnson, E. M. Induction of BIM, a proapoptotic BH3-only BCL-2 family
member, is critical for neuronal apoptosis. Neuron, 29: 615– 628, 2001.
44. Blagosklonny, M. V. Unwinding the loop of Bcl-2 phosphorylation. Leukemia
(Baltimore), 15: 869 – 874, 2001.
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Downloaded from cancerres.aacrjournals.org on July 8, 2017. © 2002 American Association for Cancer Research.
Epothilone B Analogue (BMS-247550)-mediated Cytotoxicity
through Induction of Bax Conformational Change in Human
Breast Cancer Cells
Hirohito Yamaguchi, Shanthi R. Paranawithana, Michael W. Lee, et al.
Cancer Res 2002;62:466-471.
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